The present invention relates to a method of determination of serum triglycerides according to an enzymatic reaction and a reagent used therefor.
The determination of triglycerides is popular as one of lipid tests for hyper-lipoidemia and clinical significance thereof is now being increased. However, the conventional methods of the determination are troublesome in operations and therefore inconvenient for usual tests. For example, in acetylacetone procedure which is most commonly employed at present, triglycerides are first isolated by extraction with an organic solvent, because the neutral fats must not be influenced by phospholipids or saccharides contained in the serum. This procedure is considerably complicated. Then, the triglycerides thus extracted are hydrolyzed with an alkali and glycerol formed by the hydrolysis is oxidized with an oxidizing agent such as sodium metaperiodate, formaldehyde thus formed is condensed with acetylacetone and degree of the color developed is determined by colorimetry. The operation thus comprises several steps. The above acetylacetone procedure is disclosed in, for example, M. J. Fletcher, "A colorimetric method for estimating serum triglycerides" Clinica Chimica Acta 22 (1968) 393-397.
Recently, a method wherein triglycerides are decomposed according to an enzymatic reaction and glycerol thus obtained is determined by increase or decrease of NADH has been attempted. For example, U.S. Pat. Application Ser. No. 98,904 filed on Dec. 16, 1970 discloses a method of analyzing a glycerol ester with a combination of lipase and protease. Further, K. ONOBU, et al. reported the determination of serum triglycerides with lipoprotein lipase-glycerol dehydrogenase in "Study of enzymatic analysis in clinical chemistry " Report 12 [Nihon Yakugaku-Kai 93 nen-Kai (1973)]. According to those methods wherein an enzymatic reaction is utilized, the extraction and adsorption steps necessitated in the acetylacetone method can be omitted. Particularly, the latter hydrolysis of triglycerides with lipoprotein lipase is advantageous in the determination of serum triglycerides, since it is selectively effective for chylomicron and lipoprotein-combinative neutral fats. The dehydrogenation reaction of glycerol is, however, disadvantageous in simplicity and rapidity, because special procedures are required, since the reaction equilibrium is inclined into the NAD formation and a wave length in ultraviolet region is used for the NADH determination. For example, in the enzymatic analysis of K. ONOBU, et al., the enzymatic dehydrogenation reaction equilibrium of glycerol with GDH is inclined into the NAD formation and, therefore, dihydroxyacetone formed by the reaction must be removed from the reaction mixture for advancing the reaction. The dehydrogenation reaction is carried out by adding hydrazine to the reaction mixture to convert dihydroxyacetone into hydrazone. Thus, at present, the determination of triglycerides according to the enzymatic reaction comprises two steps. In addition, in the determination of the resulting NADH at a wave length of 340nm, bilirubin present in the blood has influence on the absorption at the wave length of 340nm and, therefore, a serum blank is indispensable for correcting the value.